The governing hypothesis of this proposal is that the Mre11 complex preserves chromosome stability by virtue of its influence on cell cycle checkpoints and DSB metabolism during DNA replication. In Specific Aim 1, Mre11 complex functions in sister chromatid association will be assessed in S. cerevisiae and murine cells through mutagenesis focused on the Rad50 hook domain. The effect of Mre11 complex depletion on sister chromatid interaction will also be tested. The experiments in this aim will test the hypothesis that the Mre11 complex promotes sister chromatid association, and that it does so via the Rad50 hook domain. In Specific Aim 2, the functions of the Mre11 complex in vivo will be examined. In the previous funding period, we established a Rad50 hypomorphic mutant mouse, the Rad50s/s mouse that exhibits hematopoietic stem cell attrition and predisposition as a result of chronic genotoxic stress. Crosses with Rad50s/s mice that increase phenotypic severity, and thereby provide mechanistic insight will be carried out. These include crosses to Rad50+/delta to decrease Rad50s dosage, to p21 deficient mice in which increased numbers of hematopoietic stem cells are present, and to p27 deficient mice, which contain increased numbers of committed hematopoietic precursors. A cohort of aged Rad50s/delta and Rad50s/s mice, transplanted with wild type hematopoietic cells will also be established to assess pathologic outcomes in non-hematopoietic tissues. We hypothesize that these crosses will broaden the spectrum of Rad50s/s pathology and therefore reveal the mechanisms of Mre11 complex functions in vivo. We have established that Rad50s/s suppresses malignancy in Atm-/- mice. Experiments proposed in Specfic Aim 3, we will test the hypothesis that Rad50s activates an alternative DNA damage response pathway in Atm-/- mice, and thereby mitigates ATM deficiency. We will test the alternative hypothesis that suppression reflects the mitigation of chromsomal instability associated with ATM deficiency. In addition to directly addressing the Rad50s Atm-/- genetic interaction, the data obtained will provide novel and important information regarding the functional interactions of the Mre11 complex with mediators of the DNA damage response.